Module 2: MICROSCOPIC CALCULATIONS
Introduction
Two important parameters in microscopy are magnification and resolving power. Changes in
the orientation of the image that you see owe it to the optical system of the microscope.
DEFINITION OF TERMS:
Power of Resolution – is the measure for the ability to tell two points apart. It describes whether two
adjoining points can still be perceived as separate.
Magnification of a Microscope – is the product of Vobjective X Vocular
Objectives:
Scanning: 4x (40x)
LPO: 10x (100x)
HPO: 40x (400x)
OIO: 100x (1000x)
Numerical Aperture – is the sine of half the angle of the cone of light from each point of the object that
can be accepted by the objective multiplied by the index of refraction of the medium in which the object is
immersed.
The N.A. represents a performance number which can be compared to the relative aperture (f-
number) of a camera lens.
The N.A. values can be used for directly comparing the resolving powers of all types of
objectives.
The larger the N.A., the higher resolving power is.
Working Distance – refers to the distance from the cover glass to the nearest point of the objective.
Focal Depth - refers to the distance between the upper and lower limits of sharpness in the image formed
by an optical system.
As you stop down the aperture iris diaphragm, the focal depth becomes larger.
The larger the N.A. of an objective the shallower the focal depth is.
Field Number – This is a number that represents the diameter in mm of the image of the field diaphragm
that is formed by the lens in front of it.
Field of View Diameter -The actual size of the field of view in mm on the object surface.
ENLARGEMENT OR MAGNIFICATION
of a specimen is the function of a two-lens system;
the ocular lens is found in the eyepiece
objective lens is situated in a revolving nose-piece.
these lenses are separated by the body tube.
the objective lens is nearer the specimen and magnifies it, producing the real image that is
projected up into the focal plane
then magnified by the ocular lens to produce the final image.
The most commonly used microscopes are equipped with a revolving nosepiece containing four objective
lenses possessing different degrees of magnification. When these are combined with the magnification of
the ocular lens, the total or overall linear magnification of the specimen is obtained.
MAGNIFICATION
does not describe the quality of the image.
magnifying an object without good resolution is called empty magnification, as the image appears
larger but no greater detail can be seen.
the microscope magnification depends on both ocular lens and objective lens magnifications.
Microscope magnification = Vobjective X Vocular
For example, the magnification of ocular lens is 10X with magnification of lenses of objectives as
follows: 4X, 10X, 40X, and 100X. So what will be the magnifications at low, medium, high power and oil
immersion?
magnification at low power = 10 x 4 = 40 times
magnification at medium power = 10 x 10 = 100 times
magnification at high power = 10 x 40 = 400 times
magnification at oil immersion type of power = 10 x 100 = 1,000 times
HOW DO YOU NOW CALCULATE THE FIELD OF VIEW IN YOUR MICROSCOPE:
1. If using an eyepiece only?
Please check the specifications of the eyepiece, let's say you have WF 10X/20. The magnification of
the eyepiece is 10X with a field number of 20. Therefore the FOV here is 10 divided by 20 is 0.5 or an
FOV diameter of 0.5 millimeters.
2. If eyepiece and objective lens are used?
Let us use the specification of the eyepiece in the example above, WF 10X/20 and you set the
objective lens at 40.
Multiple 10 (Vocular ) by 40 (Vobjective ) to get 400. An FOV diameter of 0.05 mm will be obtained in
dividing 20 by 400.
If you know the dFOV for low power lens, FOV of higher power lenses can be calculated using the
ratio as follows and vice versa:
OBJECTIVE DIAMETER OF FIELD OF MAGNIFICATION (10x
VIEW ocular)
4x 4.0 mm (4.45) 40x
10x 2.0 mm (1.78) 100x
40x 0.4 mm (0.45) 400x
100x 0.2 mm (0.178 100x
Low Power Magnification × Low Power Field of View = Higher Power Magnification ×
Higher Power Field of View
Example: The Vocular of 10× with the Vobjective of 10× produced the field of view of 800 µm. Compute for
the field of view when the Vocular is 40× and the Vobjective is 100×.
Solution:
The magnification at lower power is 10 × 10 = 100
The magnification at higher power is 40 × 100 = 4000
Field of view = 100 × 800 / 4000 = 20 µm
LESSON 2: OBJECT (ACTUAL) SIZE
Using medium power (10x) objective, the diameter of the field of view is measured as 2mm.
Looking across the diameter are cells aligned. Counting it gives you 8 cells.
Therefore there are 8 cells visible across the dFOV of 2 mm.
You are determining the approximate size of each cell, dividing the
dFOV by number of cells (2 mm / 8 ) will give you 0.25 mm.
The size of each cell is therefore 0.25 mm or 250 µm.
LESSON 3: DRAWING MAGNIFICATION
Below is the equation for calculating the drawing magnification.
size of drawn object in μm
Magnification of drawing = ---------------------------------------
object’s actual size in μm
Let us say the drawing is roughly 7 cm long. This equates to 70 mm or 70,000µm. The actual size of
the specimen is about 500 µm. What is the drawing magnification?
Drawing magnification = drawing size / actual size.
70,000 µm / 500 µm = 140 X
Drawing Magnification = 140 X
All unknown sizes should be expressed in millimeters (mm) round off to 2 decimal places.
Actual Specimen Size Image (Drawing) Magnificatio
Size n
1 5 μm 2 cm
2 25 μm 200
3 5 cm 10x
4 0.5mm 25x
5 5 mm 10 cm
6 10 mm 365x
7 100 μm 15 mm
8 1 cm 3x
9 40 μm 20x